Figure 5. The synergistic effect of anionic phospholipids in DOPC/CH membranes on the photoincorporation of [¹²⁵I]TID into subunits of the nAChR.

Affinity-purified nAChRs were reconstituted into lipid vesicles comprised of DOPC/DOPS/DOPI/DOPA/CH at a molar ratio of 1.5:0.3:0.15:0.05:1 (that is 50% PC/ 10% PS/ 5% PI/ 1.6% PA/ 33% CH). The reconstituted membranes were equilibrated for 1 h with [¹²⁵I]TID (0.4 μM) in the absence (− lanes) and in the presence (+ lanes) of 400 μM Carb, irradiated at 365 nm for 7 min, and the polypeptides resolved by SDS-PAGE. A, corresponding autoradiograph of SDS-PAGE gel containing [¹²⁵I]TID labeled nAChRs reconstituted into DOPC/DOPS/DOPI/DOPA/CH membranes. The positions of the nAChR subunits are indicated on the left. B, Labeled nAChR subunit bands were excised and the amount of [¹²⁵I]TID photoincorporated into each subunit determined by γ counting (5 min of counting time). [¹²⁵I]TID incorporation into γ' was added to that of the γ-subunit. Shown are bar graphs of the amount of ¹²⁵I cpm incorporated into each nAChR subunit in the absence or presence of carbamylcholine (−/+) and presented as the average of triplicate determinations (error bars indicate the standard error). In the absence of agonist, the ratio of [¹²⁵I]TID labeling in the γ– and α–subunit was calculated (γ/α ratio = 4.6).